Antifibrotic activity of GAS6 inhibitor

ABSTRACT

Compositions and methods are provided for treating fibrosis in a mammal by administering a therapeutic dose of a pharmaceutical composition that inhibits AXL, MER or Tyro3 protein activity, for example by inhibition of the binding interaction between AXL, MER or Tyro3 and its ligand GAS6.

CROSS REFERENCE

This application is a 371 application and claims the benefit of PCTApplication No. PCT/US2015/066498, filed Dec. 17, 2015, which claimsbenefit of U.S. Provisional Patent Application No. 62/093,937, filedDec. 18, 2014, which applications are incorporated herein by referencein their entirety.

BACKGROUND OF THE INVENTION

Fibrosis, defined by the excessive accumulation of extracellular matrixcomponents (ECM) in and around inflamed or damaged tissue, is associatedwith several inflammatory conditions. In these situations, normal tissuerepair response turns into an irreversible fibrotic response throughdysregulation of response to stress or injury. Fibrosis can lead topermanent scarring, organ malfunction and, ultimately, death, as seen inend-stage liver disease, kidney disease, idiopathic pulmonary fibrosis(IPF), retinal fibrosis, and heart failure from cardiac fibrosis.Fibrosis also influences tumor invasion and metastasis, chronic graftrejection and the pathogenesis of many progressive myopathies.

Many distinct triggers can contribute to the development of progressivefibrotic disease, but regardless of the initiating events, a commonfeature is the activation of ECM-producing myofibroblasts, which are thekey mediators of fibrotic tissue remodeling. Many elements of the innateand adaptive immune response participate in the differentiation andactivation of fibroblasts. During equilibrium, tissue-residentfibroblasts are quiescent. To repair tissues after injury, thesetissue-resident fibroblasts are activated and transformed intomyofibroblasts. Myofibroblasts secrete large amounts of ECM, aiding incontracture and closure and orchestrating many aspects of the healingresponse. Myofibroblast activation, proliferation and survival aremediated by a variety of secreted, soluble and physical factors in themilieu, such as cytokines including IL-1, TNF, TGF-β1 and IL-13, growthfactors such as CTGF and PDGF, and matrix factors such as hyaluronanfragments, mechanical stress and stiffness. During normal wound healing,myofibroblasts undergo apoptosis after re-epithelialization of thewound, but myofibroblasts in fibrotic loci are resistant to programmedcell death. Pathways that elicit and recruit high numbers ofmyofibroblasts and those that engender resistance to apoptosis areactive areas of fibrosis research.

Because ECM-producing myofibroblasts are the final common pathogeniccell in fibrotic diseases, any therapy that successfully ablates theiractivity could have broad antifibrotic activity. Targeting keyinflammatory pathways may also be useful in the treatment of fibrosis.Because TNF-α has emerged as a key driver of fibrosis in manyexperimental studies, clinical trials have been initiated to examinewhether inhibitors of the TNF α pathway could be used to treat IPF andother scarring disorders.

Antifibrotic compositions and methods of use thereof are of greatclinical and humanitarian interest. The present invention addresses thisneed.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful forinhibiting fibrosis via inhibition of AXL and/or GAS6 related pathways.In some embodiments the inhibitor is a high affinity soluble AXL variantpolypeptide. In some embodiments the fibrosis is associated with cancerand tumor growth, i.e. tumor related tissue fibrosis, including withoutlimitation pancreatic cancer. In other embodiments the fibrosis isassociated with chronic inflammation or injury in other tissues,including without limitation liver, lung, kidney, and the like.

In some embodiments, the inhibitor is a polypeptide, a polynucleotide, asmall molecule, an antibody, an antibody fragment or antibodydrug-conjugate capable of binding to GAS6 with increased affinitycompared to wild-type AXL, MER or Tyro3. In some embodiments, theinhibitor agent binds to two or more epitopes on a single GAS6. In someembodiments, the inhibitor agent is capable of binding to the major andminor AXL, MER or Tyro3 binding sites on a single GAS6. In someembodiments, the inhibitor agent is capable of binding the major AXL,MER or Tyro3 binding site of GAS6 and one or more additional GAS6epitopes on a single GAS6. In some embodiments, the inhibitor agent iscapable of binding to the minor AXL, MER or Tyro3 binding site on GAS6and one or more additional epitopes on a single GAS6. In someembodiments, the inhibitor agent is capable of binding two or moreepitopes on a single GAS6. In some embodiments, the inhibitor agent iscapable of antagonizing the major and/or minor GAS6/receptor bindinginteraction, and wherein the receptor is selected from AXL, MER andTyro3. In some embodiments, the inhibitor agent is capable ofantagonizing the major GAS6/receptor binding interaction, and whereinthe receptor is selected from AXL, MER and Tyro3. In some embodiments,the inhibitor agent is capable of antagonizing the minor GAS6/receptorbinding interaction, and wherein the receptor is selected from AXL, MERand Tyro3. In some embodiments the inhibitor is a small molecule thatbinds to the kinase domain of AXL and inhibit the intracellularsignaling of AXL activation.

In some embodiments, the inhibitor agent is a small molecule,polypeptide, a polypeptide-carrier fusion, a polypeptide-Fc fusion,polypeptide-conjugate, a polypeptide-drug conjugate, an antibody, abispecific antibody, an antibody drug conjugate, an antibody fragment,an antibody-related structure, or a combination thereof. In someembodiments, the inhibitor agent is a natural or synthetic polypeptide.In some embodiments, the inhibitor agent is a non-antibody polypeptide.

In some embodiments, the inhibitor agent is a darpin, an avimer, anadnectin, an anticalin, an affibody, a maxibody or a combinationthereof. In some embodiments, the inhibitor agent is apolypeptide-conjugate or an antibody-conjugate. In some embodiments, theinhibitor agent comprises a polypeptide-polymer conjugate, and whereinthe polymer is a PEG, a PEG-containing polymer, a degradable polymer, abiocompatible polymer or a hydrogel.

In some embodiments, the inhibitor agent is a polypeptide, wherein thepolypeptide comprises a soluble AXL variant polypeptide wherein the AXLpolypeptide lacks the AXL transmembrane domain and has at least onemutation relative to wild-type that increases affinity of the AXLpolypeptide binding to GAS6 compared to wild-type AXL.

In some embodiments, the inhibitor agent is a polypeptide, wherein thepolypeptide comprises a soluble MER variant polypeptide wherein said MERpolypeptide lacks the MER transmembrane domain and has at least onemutation relative to wild-type that increases affinity of the MERpolypeptide binding to GAS6 compared to wild-type MER.

In some embodiments, the inhibitor agent is a polypeptide, wherein saidpolypeptide comprises a soluble Tyro3 variant polypeptide wherein saidTyro3 polypeptide lacks the Tyro3 transmembrane domain and has at leastone mutation relative to wild-type that increases affinity of the Tyro3polypeptide binding to GAS6 compared to wild-type Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide inhibitsbinding between a wild-type AXL, MER and/or Tyro3 polypeptide and a GAS6protein in vivo or in vitro.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks afunctional fibronectin (FN) domain and/or wherein said AXL, MER or Tyro3variant polypeptide exhibits increased affinity of the polypeptidebinding to GAS6 compared to wild-type AXL, MER or Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain, has more than one Ig1 domain and wherein said AXL,MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL,MER or Tyro3 variant polypeptide binding to GAS6 compared to wild-typeAXL, MER or Tyro3.

In some embodiments, the polypeptide has two Ig1 domains. In someembodiments, the polypeptide has three Ig1 domains. In some embodiments,the soluble AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain, has more than one Ig2 domain and wherein said AXL,MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL,MER or Tyro3 polypeptide binding to GAS6 compared to wild-type AXL, MERor Tyro3. In some embodiments, the polypeptide has two Ig2 domains.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 transmembrane domain, has morethan one Ig1 domain, more than one Ig2 domain, and wherein said AXL, MERor Tyro3 variant polypeptide exhibits increased affinity of the AXL, MERor Tyro3 variant polypeptide binding to GAS6 compared to wild-type AXL,MER or Tyro3.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 transmembrane domain, lacks afunctional fibronectin (FN) domain, has more than one Ig1 domain, morethan one Ig2 domain, and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3 variantpolypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3.

In some embodiments, the soluble AXL, MER or Tyro3 variant polypeptidehas two Ig1 domains and two Ig2 domains. In some embodiments, theimmunoglobulin domains are connected directly. In some embodiments, theimmunoglobulin domains are connected indirectly. In some embodiments,the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide,wherein said variant polypeptide lacks the AXL, MER or Tyro3transmembrane domain, is capable of binding both the major and minorbinding site of a single GAS6 and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3polypeptide binding to GAS6.

In some embodiments, the polypeptide has one Ig1 domain and lacks afunctional Ig2 domain. In some embodiments, the polypeptide is a solubleAXL, MER or Tyro3 variant polypeptide, wherein said soluble AXL, MER orTyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembranedomain, has one Ig1 domain, lacks a functional Ig2 domain and whereinsaid AXL, MER or Tyro3 variant polypeptide exhibits increased affinityof the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared towild-type AXL, MER or Tyro3.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 transmembrane domain, lacks afunctional fibronectin (FN) domain, has one Ig1 domain, lacks afunctional Ig2 domain and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3 variantpolypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide is afusion protein comprising an Fc domain. In some embodiments, the variantpolypeptide lacks the AXL, MER or Tyro3 intracellular domain. In someembodiments, the soluble AXL, MER or Tyro3 variant polypeptide furtherlacks a functional fibronectin (FN) domain and wherein said variantpolypeptide exhibits increased affinity of the polypeptide binding toGAS6. In some embodiments, the soluble AXL, MER or Tyro3 variantpolypeptide comprises at least one amino acid modification relative tothe wild-type AXL, MER or Tyro3 sequence.

In some embodiments, the soluble AXL variant polypeptide comprises atleast one amino acid modification within a region selected from thegroup consisting of 1) between 15-50, 2) between 60-120, and 3) between125-135 of the wild-type AXL sequence (SEQ ID NO:1).

In some embodiments, the soluble AXL variant polypeptide comprises atleast one amino acid modification at position 19, 23, 26, 27, 32, 33,38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105, 109,112, 113, 116, 118, or 127 of the wild-type AXL sequence (SEQ ID NO: 1)or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide comprises atleast one amino acid modification selected from the group consistingof 1) A19T, 2) T23M, 3) E26G, 4) E27G or E27K 5) G32S, 6) N33S, 7) T38I,8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14) V79M, 15)Q86R, 16) D87G, 17) D88N, 18) 190M or 190V, 19) V92A, V92G or V92D, 20)197R, 21) T98A or T98P, 22) T105M, 23) Q109R, 24) V112A, 25) F113L, 26)H116R, 27) T118A, 28) G127R or G127E, and 29) G129E and a combinationthereof.

In some embodiments, the AXL variant polypeptide comprises amino acidchanges relative to the wild-type AXL sequence (SEQ ID NO: 1) at thefollowing positions: (a) glycine 32; (b) aspartic acid 87; (c) valine92; and (d) glycine 127.

In some embodiments, the AXL variant polypeptide comprises amino acidchanges relative to the wild-type AXL sequence (SEQ ID NO: 1) at thefollowing positions: (a) aspartic acid 87 and (b) valine 92.

In some embodiments, the AXL variant polypeptide comprises amino acidchanges relative to the wild-type AXL sequence (SEQ ID NO: 1) at thefollowing positions: (a) glycine 32; (b) aspartic acid 87; (c) valine92; (d) glycine 127 and (e) alanine 72.

In some embodiments, the AXL variant polypeptide comprises amino acidchanges relative to the wild-type AXL sequence (SEQ ID NO: 1) at thefollowing position: alanine 72.

In some embodiments, in the AXL variant polypeptide glycine 32 residueis replaced with a serine residue, aspartic acid 87 residue is replacedwith a glycine residue, valine 92 residue is replaced with an alanineresidue, or glycine 127 residue is replaced with an arginine residue ora combination thereof.

In some embodiments, in the AXL variant polypeptide aspartic acid 87residue is replaced with a glycine residue or valine 92 residue isreplaced with an alanine residue or a combination thereof.

In some embodiments, in the AXL variant polypeptide alanine 72 residueis replaced with a valine residue.

In some embodiments, in the AXL variant polypeptide glycine 32 residueis replaced with a serine residue, aspartic acid 87 residue is replacedwith a glycine residue, valine 92 residue is replaced with an alanineresidue, glycine 127 residue is replaced with an arginine residue or analanine 72 residue is replaced with a valine residue or a combinationthereof.

In some embodiments, the AXL variant comprises amino acid changesrelative to the wild-type AXL sequence (SEQ ID NO: 1) at the followingpositions: (a) glutamic acid 26; (b) valine 79; (c) valine 92; and (d)glycine 127.

In some embodiments, in the AXL variant polypeptide glutamic acid 26residue is replaced with a glycine residue, valine 79 residue isreplaced with a methionine residue, valine 92 residue is replaced withan alanine residue, or glycine 127 residue is replaced with an arginineresidue or a combination thereof.

In some embodiments, in the AXL variant polypeptide comprises at leastan amino acid region selected from the group consisting of amino acidregion 19-437, 130-437, 19-132, 21-121, 26-132, 26-121 and 1-437 of thewild-type AXL polypeptide (SEQ ID NO: 1), and wherein one or more aminoacid modifications occur in said amino acid region.

In some embodiments, in the AXL variant polypeptide comprises amino acidchanges relative to the wild-type AXL sequence (SEQ ID NO: 1) at thefollowing positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine72; and valine 92.

In some embodiments, in the AXL variant polypeptide glycine 32 isreplaced with a serine residue, aspartic acid 87 is replaced with aglycine residue, alanine 72 is replaced with a valine residue, andvaline 92 is replaced with an alanine residue, or a combination thereof.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain and wherein said AXL variant comprises aminoacid changes relative to wild-type AXL sequence (SEQ ID NO:1) at thefollowing positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine72; and (d) valine 92.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain and wherein glycine 32 is replaced with a serineresidue, aspartic acid 87 is replaced with a glycine residue, alanine 72is replaced with a valine residue, and valine 92 is replaced with analanine residue, or a combination thereof.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain and wherein said AXL variant comprises aminoacid changes relative to wild-type AXL sequence (SEQ ID NO:1) at thefollowing positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine72; (d) valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain and wherein glycine 32 is replaced with a serineresidue, aspartic acid 87 is replaced with a glycine residue, alanine 72is replaced with a valine residue, valine 92 is replaced with an alanineresidue, and glycine 127 is replaced with an arginine residue or acombination thereof.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain, lacks a functional FN domain, and wherein saidAXL variant comprises amino acid changes relative to wild-type AXLsequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b)aspartic acid 87; (c) alanine 72; and (d) valine 92.

In some embodiments, the soluble AXL variant is a fusion proteincomprising an Fc domain, lacks a functional FN domain, and whereinglycine 32 is replaced with a serine residue, aspartic acid 87 isreplaced with a glycine residue, alanine 72 is replaced with a valineresidue, and valine 92 is replaced with an alanine residue, or acombination thereof.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain, lacks a functional FN domain, and wherein saidAXL variant comprises amino acid changes relative to wild-type AXLsequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b)aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL variant is a fusion proteincomprising an Fc domain, lacks a functional FN domain, and whereinglycine 32 is replaced with a serine residue, aspartic acid 87 isreplaced with a glycine residue, alanine 72 is replaced with a valineresidue, valine 92 is replaced with an alanine residue, and glycine 127is replaced with an arginine residue or a combination thereof.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain, lacks a functional FN domain, lacks an Ig2domain, and wherein said AXL variant comprises amino acid changesrelative to wild-type AXL sequence (SEQ ID NO:1) at the followingpositions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72 and (d)valine 92.

In some embodiments, the soluble AXL variant is a fusion proteincomprising an Fc domain, lacks a functional FN domain, lacks an Ig2domain and wherein glycine 32 is replaced with a serine residue,aspartic acid 87 is replaced with a glycine residue, alanine 72 isreplaced with a valine residue, and valine 92 is replaced with analanine residue or a combination thereof.

In some embodiments, the soluble AXL polypeptide is a fusion proteincomprising an Fc domain, lacks a functional FN domain, lacks an Ig2domain, and wherein said AXL variant comprises amino acid changesrelative to wild-type AXL sequence (SEQ ID NO:1) at the followingpositions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d)valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL variant is a fusion proteincomprising an Fc domain, lacks a functional FN domain, lacks an Ig2domain and wherein glycine 32 is replaced with a serine residue,aspartic acid 87 is replaced with a glycine residue, alanine 72 isreplaced with a valine residue, valine 92 is replaced with an alanineresidue, and glycine 127 is replaced with an arginine residue or acombination thereof.

In some embodiments, the soluble AXL variant polypeptide has an affinityof at least about 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M or 1×10⁻¹² Mfor GAS6.

In some embodiments, the soluble AXL variant polypeptide exhibits anaffinity to GAS6 that is at least about 5-fold stronger, at least about10-fold stronger or at least about 20-fold stronger than the affinity ofthe wild-type AXL polypeptide.

In some embodiments, the soluble AXL, MER or Tyro3 variant polypeptidefurther comprises a linker. In some embodiments, the linker comprisesone or more (GLY)₄SER units (SEQ ID NO: 4). In some embodiments, thelinker comprises 1, 2, 3 or 5 (GLY)₄SER units (SEQ ID NO: 4).

In some embodiments, the soluble AXL MER and/or Tyro3 variantpolypeptide inhibits binding between wild-type AXL, MER and/or Tyro3polypeptide and a GAS6 protein in vivo or in vitro.

In some embodiments, the soluble AXL variant polypeptide is a fusionpolypeptide comprising an Fc domain.

Thus, the invention relates to an inhibitor of AXL and/or GAS6 relatedpathways for use for preventing or treating fibrosis, comprisingadministering to an individual in need thereof a therapeuticallyeffective amount of an inhibitor of the invention.

Also provided is a pharmaceutical composition comprising an inhibitor ofAXL and/or GAS6 related pathways as described above, and additionally atleast one other antifibrotic compound; or if appropriate in combinationwith a chemotherapeutic drug for the treatment of cancer. Also providedis the use of an inhibitor of the invention for the manufacture of amedicament for preventing or treating fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following FIGURES.

FIG. 1. Analysis of pancreatic cancer fibrosis after treatment withinhibitory axl polypeptide.

DEFINITIONS

In the description that follows, a number of terms conventionally usedin the field of cell culture are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given to such terms, the followingdefinitions are provided.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of two or more amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. The terms“antibody” and “antibodies” are used interchangeably herein and refer toa polypeptide capable of interacting with and/or binding to anothermolecule, often referred to as an antigen. Antibodies can include, forexample “antigen-binding polypeptides” or “target-molecule bindingpolypeptides.” Antigens of the present invention can include for exampleany polypeptides described in the present invention.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. All single lettersused in the present invention to represent amino acids are usedaccording to recognized amino acid symbols routinely used in the field,e.g., A means Alanine, C means Cysteine, etc. An amino acid isrepresented by a single letter before and after the relevant position toreflect the change from original amino acid (before the position) tochanged amino acid (after position). For example, A19T means that aminoacid alanine at position 19 is changed to threonine.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” thus encompass individuals havingcancer, including without limitation, pancreatic cancer, adenocarcinomaof the ovary or prostate, breast cancer, glioblastoma, etc., includingthose who have undergone or are candidates for resection (surgery) toremove cancerous tissue. Subjects may be human, but also include othermammals, particularly those mammals useful as laboratory models forhuman disease, e.g. mouse, rat, etc.

The definition of an appropriate patient sample encompasses blood andother liquid samples of biological origin, solid tissue samples such asa biopsy specimen or tissue cultures or cells derived there from and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas endometrial cells, kidney disease cells, inflammatory disease cellsand/or transplant rejection (GVHD) cells. The definition also includessample that have been enriched for particular types of molecules, e.g.,nucleic acids, polypeptides, etc. The term “biological sample”encompasses a clinical sample, and also includes tissue obtained bysurgical resection, tissue obtained by biopsy, cells in culture, cellsupernatants, cell lysates, tissue samples, organs, bone marrow, blood,plasma, serum, and the like. A “biological sample” includes a sampleobtained from a patient's sample cell, e.g., a sample comprisingpolynucleotides and/or polypeptides that is obtained from a patient'ssample cell (e.g., a cell lysate or other cell extract comprisingpolynucleotides and/or polypeptides); and a sample comprising samplecells from a patient. A biological sample comprising a sample cell froma patient can also include normal, non-diseased cells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of fibrosis.

Fibrosis is the excessive accumulation of extracellular matrixcomponents (ECM) in and around inflamed or damaged tissue, oftenassociated with chronic inflammation or cancer. The presence of fibrosiscan be detected by means known in the art, for example by examination oftissue for excess scarring. Prior to fibrosis, an individual may bedetermined to be susceptible based on undesirable increase ininflammatory mediators that can exacerbate tissue injury, such as IL-1β,TNF-α and reactive oxygen and nitrogen species. Profibrotic mediatorssuch as TGF-β1 may be present. Also present are activatedmyofibroblasts, which may be resistant to induction of apoptosis.

Exemplary forms of fibrosis include, but are not limited to, tumorfibrosis, cardiac fibrosis, liver fibrosis, kidney fibrosis, lungfibrosis, dermal scarring and keloids, and Alzheimer's disease. In stillfurther embodiments, cardiac fibrosis is associated with hypertension,hypertensive heart disease (HHD), myocardial infarction (MI), cardiacscarring related to ischemia congestive heart failure, cardiomyopathy,post-myocardial infarction defects in heart function, atherosclerosis,and restenosis. Kidney fibrosis may include, but not be limited to,diabetic nephropathy, vesicoureteral reflux, tubulointerstitial renalfibrosis, glomerulonephritis or glomerular nephritis (GN), focalsegmental glomerulosclerosis, membranous glomerulonephritis, ormesangiocapillary GN. Liver fibrosis may include, but not be limited to,cirrhosis, and associated conditions such as chronic viral hepatitis,non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis(ASH), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis(PBC), biliary cirrhosis, autoimmune hepatitis). Lung fibrosis mayinclude idiopathic pulmonary fibrosis (IPF) or cryptogenic fibrosingalveolitis, chronic fibrosing interstitial pneumonia, interstitial lungdisease (ILD), and diffuse parenchymal lung disease (DPLD)), lungscarring including without limitation damage from bacterial viral orfungal infection, emphysema, chronic obstructive pulmonary disease(COPD); and chronic asthma may also be prevented, treated, orameliorated with compositions of described herein. Also included isglaucoma; age-related macular degeneration (wet AMD and dry AMD).

Renal fibrosis is the consequence of an excessive accumulation ofextracellular matrix that occurs in virtually every type of chronickidney disease. The pathogenesis of renal fibrosis is a progressiveprocess that ultimately leads to end-stage renal failure, a devastatingdisorder that requires dialysis or kidney transplantation. In asimplistic view, renal fibrosis represents a failed wound-healingprocess of the kidney tissue after chronic, sustained injury. Severalcellular pathways, including mesangial and fibroblast activation as wellas tubular epithelial-mesenchymal transition, have been identified asthe major avenues for the generation of the matrix-producing cells indiseased conditions.

Pulmonary fibrosis is characterized by lung inflammation and abnormaltissue repair, resulting in the replacement of normal functional tissuewith an abnormal accumulation of fibroblasts and deposition of collagenin the lung. This process involves cellular interactions via a complexcytokine-signaling mechanism and heightened collagen gene expression,ultimately resulting in its abnormal collagen deposition in the lung. Inaddition to inflammatory cells, the fibroblast and signaling events thatmediate fibroblast proliferation and myofibroblasts play important rolesin the fibrotic process. However, the most potent anti-inflammatorydrugs that have been widely used in the treatment of pulmonary fibrosisdo not seem to interfere with the fibrotic disease progression.

Hepatic fibrosis is an accumulation in the liver of connective tissue inresponse to hepatocellular damage of nearly any cause. It results fromexcessive production or deficient degradation of the extracellularmatrix. Fibrosis itself causes no symptoms but can lead to portalhypertension or cirrhosis.

Systemic sclerosis is a chronic disease of unknown cause characterizedby diffuse fibrosis, degenerative changes, and vascular abnormalities inthe skin, joints, and internal organs (especially the esophagus, lowerGI tract, lung, heart, and kidney). Common symptoms include Raynaud'sphenomenon, polyarthralgia, dysphagia, heartburn, and swelling andeventually skin tightening and contractures of the fingers. Lung, heart,and kidney involvement accounts for most deaths. Specific treatment isdifficult, and emphasis is often on treatment of complications.

A variety of drugs have been tried in various fibroses, particularlylung fibrosis, with very little success. Anti-inflammatory drugsincluding prednisolone and azathioprine have little effect on fibrosissuggesting that inflammation is only the initiator, but not the driverof the disease. The use of non-specific anti-proliferatives likecolchicine and cyclophosphamide will also prevent repair of the fibrotictissue by impairing e.g. epithelial growth. Treatment with IFN-γ hasshown some utility but is limited by severe side effects.

By the time a typical patient presents with fibrosis-related symptoms(e.g. difficulty breathing for lung fibrosis, cirrhosis for liverfibrosis, etc.), the fibrosis in the target organ is often quite severe,with much of the target organ architecture having been replaced withextracellular matrix. Stopping this ongoing fibrosis can extend lifespanand improve quality of life. Areas of the target organ where thefibrosis is not extensive may be restored to normal architecture withsuitable treatment.

In some embodiments, tumor fibrosis is associated with pancreaticcancer. Pancreatic cancer is characterized by a prominentdesmoplastic/stromal reaction. Pancreatic stellate cells (PSCs) are theprincipal source of fibrosis in the stroma and interact closely withcancer cells to create a tumor facilitatory environment that stimulateslocal tumor growth and distant metastasis. Pancreatic fibrosis isinitiated when PSCs become activated and undergo morphological andfunctional changes, so that the rate of extracellular matrix (ECM)deposition exceeds the rate of ECM degradation in the gland. It is nowwell established that pancreatic cancer cells activate PSCs leading toincreased fibrosis. There is significant evidence showing that theintense stromal/desmoplastic reaction around tumor elements (a featureof the majority of pancreatic cancers) plays an important role in tumorprogression.

A key histopathological feature of pancreatic cancer which is associatedwith its innate clinical and biological aggressiveness is itsdesmoplastic (stromal) reaction. Stroma production is stimulated bycancer-cell derived growth factors including transforming growthfactor-β (TGFβ), hepatocyte growth factor (HGF), fibroblast growthfactor (FGF), insulin-like growth factor 1 (IGF-1) and epidermal growthfactor (EGF). The desmoplastic reaction is composed of extracellularmatrix (ECM) proteins, primarily type I and III collagen, fibronectinand proteoglycans; small endothelium lined vessels; and a diversepopulation of cells including inflammatory cells, fibroblasts andstellate cells. The stroma can form up to 90% of the tumor volume, aproperty which is unique to pancreatic cancer. The tumormicroenvironment in pancreatic cancer plays a role in itschemoresistance.

While stromal cells do not exhibit the genetic transformations seen inmalignant pancreatic cancer cells, they are altered by cytokines andgrowth factors secreted by inflammatory cells and tumor cells.Reciprocally, the stromal cells promote tumor cell migration, growth,invasion and resistance to drugs and apoptosis. Staining pancreaticcancer tissue sections of patients for alpha smooth muscle actin (α-SMAthe cytoskeletal protein marker for PSC activation) and collagen shows ahigh activated stroma index (α-SMA/collagen) correlated with a poorprognosis. The extensive ECM deposition by PSCs in pancreatic cancercauses distortion and compression of tumor vasculature by fibrous tissuewhich contributes to tumor hypoxia, a determinant of chemoresistance.

“Inhibitors,” “activators,” and “modulators” of AXL, MER or Tyro3 or itsligand GAS6 are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor receptor or ligand binding or signaling, e.g., ligands, receptors,agonists, antagonists, and their homologs and mimetics.

By “inhibitor” is meant an agent that is able to reduce or to abolishthe interaction between GAS6 and a TAM receptor. Preferably, saidinhibitor is able to reduce or to abolish the interaction by at least10, 20, 30, 40%, more preferably by at least 50, 60, 70%, and mostpreferably by at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%.Included are polypeptides including without limitation antibodies,polynucleotides, small molecules and the like that act to inhibit theGAS6 and/or AXL signaling pathways.

Non limiting examples of small molecule inhibitors include ONO-9330547,which is a small molecule inhibitor that binds to Axl and Mer kinasewith a potency (IC) of 2.2 and 0.4 nM, see ASH abstracts 2014, #999. TheAxl-specific inhibitor, TP-0903 (Tolero Pharmaceuticals), see ASHabstracts 2014, #2350 may be used alone or in combination with FLT3inhibitor PKC412 (Park et al, Blood 121, 2064-73, 2013).

Inhibitors of interest also include LY2801653 (Eli Lilly and Co) Journalof the American Association for Cancer Research. 2013; 19:5699-5710.Investigational new drugs. 2013; 31:833-844; MP-470 or Amuvatinib (AstexPharm). Oncogene. 2007; 26:3909-3919; SKI-606, PF-5208763, or Bosutinib,(Pfizer), Carcinogenesis. 2014; 35:769-775; MGCD 265 (Mirati). Journalof Clinical Oncology. 2010; 28:e13595. European journal of cancer. 2012;48:95-96; MGCD 516 (Mirati). Poster: #6130 Proceedings: AACR 104thAnnual Meeting 2013; Apr. 6-10, 2013; (Washington, D.C.); ASP2215(Astellas) J Clin Oncol 32:5s, 2014 (suppl; abstr 7071), J Clin Oncol32:5s, 2014 (suppl; abstr 7070), Blood. 2013; 121:2064-2073;XL184/Cabozanitinib (Exelixis). Molecular cancer therapeutics. 2011;10:2298-2308; BMS 777607 or ASLAN 002. Journal of medicinal chemistry52, 1251-1254 (2009); GSK163089/XL880 or Foretinib (GSK). Cancerresearch. 2009; 69:6871-6878; SGI-7079 (Astex Pharma) Cancer research.2013; 73:6516-6525., Clinical cancer research: 2013; 19:279-290; S49076(Servier) Molecular cancer therapeutics. 2013; 12:1749-1762;R428/BGB324, (BergenBio). Oncogene. 1997; 14:2619-2631. Naturebiotechnology. 2013; 31:775-776; DP3975 (Deciphera Biotech). Oncogene.2011; 30:1643-1652; NPS-1034 (NeoPharma). Cancer research. 2014;74:253-262; LDC 126. Nature. 2014; 507:508-512; NA80x1. Cancer research.2008; 68:1905-1915; PF-2341066/Crizotinib (Pfizer) Nature biotechnology29, 1046-1051 (2011), ACS medicinal chemistry letters 2, 907-912 (2011);Vandetinib. Nature biotechnology 29, 1046-1051 (2011); Sunitinib.(Pfizer). Nature biotechnology 29, 1046-1051 (2011). Br J Cancer 101,1717-1723 (2009); Lestaurtinib/CEP-701. Nature biotechnology 29,1046-1051 (2011); CEP-40783 (Teva). Abstract #C272, EORTC-AACR Oct.19-23, 2013, Abstract #C275, EORTC-AACR Oct. 19-23, 2013; Neratinib.Nature biotechnology 29, 1046-1051 (2011); AT9283. Journal of medicinalchemistry 52, 379-388 (2009); R406. Nature biotechnology 29, 1046-1051(2011); MK-2461. Journal of medicinal chemistry 52, 1251-1254 (2009);SU-14813. Nature biotechnology 29, 1046-1051 (2011); BMS-796302. Naturereviews. Clinical oncology 9, 314-326 (2012); JNJ-28312141. Naturebiotechnology 29, 1046-1051 (2011). Molecular cancer therapeutics 8,3151-3161 (2009); Diaminopyrimidine. ACS medicinal chemistry letters 2,907-912 (2011); Warfarin. JASN (1999) vol. 10 no. 12 2503-2509; UNC569,UNC1062, UNC1666, UNC2025 described in ACS medicinal chemistry letters3, 129-134 (2012). Molecular cancer therapeutics 12, 2367-2377 (2013). JClin Invest 123, 2257-2267 (2013). European journal of medicinalchemistry 65, 83-93 (2013). Blood 122, 3849 (2013). American Associationof Cancer Researchers Annual Meeting, San Diego, Calif., Abstract #1740(2014). Journal of medicinal chemistry 57, 7031-7041 (2014).

Monoclonal antibodies that specifically target AXL have been described,including 12A11, Mab173, YW327.652 and, more recently, D9 and E8. Othersincluded Chugai monoclonal antibody to AXL, BergenBio monoclonalantibody to AXL, and Amgen's monoclonal against GAS6. Antibody mediateddecreases in cell-surface AXL expression induced apoptosis, enhancedsensitivity to chemotherapy and inhibited xenograft growth of NSCLC,pancreatic cancer and Kaposi's sarcoma. Similarly, an antibody thatrecognizes MERTK inhibited migration in glioblastoma cells and decreasedcolony-forming potential and chemoresistance of NSCLC cell lines, whichphenocopies the effects of MERTK knockdown.

Antibodies that block TYRO3-dependent signaling have also been reported.An aptamer with high affinity for AXL (with a dissociation constant (Kd)of 12 nM) has been developed and was shown to mediate inhibition of AXLactivity in NSCLC models, decreasing tumour cell migration, invasion andxenograft tumour growth. TAM RTK extracellular domains can bind toligand with high affinity and may thereby function as ‘sinks’ toeliminate free ligand. Recombinant proteins consisting of all or part ofthe extracellular domain of AXL, MERTK or TYRO3 fused to an Fc domainderived from human immunoglobulin G (IgG) have been generated, and theyinhibit both GAS6-dependent tumour cell survival in culture andmetastasis in animal models. In addition, both AXL and MERTK ligandsinks have been effective in preventing platelet aggregation and clotformation in vivo.

See, for example, Varnum et al. Nature 373, 623-626 (1995); Stitt et al.Cell 80, 661-670 (1995); Nagata et al. J. Biol. Chem. 271, 30022-30027(1996); Angelillo-Scherrer et al. J. Clin. Invest. 115, 237-246 (2005);Park et al. Blood 121, 2064-2073 (2013); Rankin et al. Proc. Natl Acad.Sci. USA 111 13373-13378 (2014); Liu et al. Blood 116, 297-305 (2010);Rogers et al. Oncogene 31, 4171-4181 (2012); Li et al. Oncogene 28,3442-3455 (2009); Ye et al. Oncogene 29, 5254-5264 (2010); Leconet etal. Oncogene (2013); Kariolis et al. Nature Chem. Biol. 10, 977-983(2014); Cummings et al. Oncotarget 26 Jun. 2014; Demarest et al.Biochemistry 52, 3102-3118 (2013); Cerchia, L. et al. Mol. Ther. 20,2291-2303 (2012); Avilla, E. et al. Cancer Res. 71, 1792-1804 (2011);Sather, S. et al. Blood 109, 1026-1033 (2007); ANTICANCER RESEARCH 34:1821-1828 (2014); Abstract 5158_AACR Apr. 13, 2013: Generation of afully human Gas6 neutralizing antibody with anti-tumor activity in vivo

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure for the purposesof obtaining an effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of effecting a partial or complete cure fora disease and/or symptoms of the disease. “Treatment,” as used herein,covers any treatment of fibrosis in a mammal, particularly in a human,and includes: (a) preventing the development of fibrosis; (b) inhibitingongoing fibrosis, i.e., arresting its development; and (c) relievingfibrosis, i.e., causing regression.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of fibrosis, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withfibrosis. The term “therapeutic effect” refers to the reduction,elimination, or prevention of the disease, symptoms of the disease, orside effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic (i.e., first therapeutic agent) and thecompounds as used herein. When administered in combination, eachcomponent can be administered at the same time or sequentially in anyorder at different points in time. Thus, each component can beadministered separately but sufficiently closely in time so as toprovide the desired therapeutic effect. First therapeutic agentscontemplated for use with the methods of the present invention includeany other agent for use in the treatment of fibrosis. Examples of suchtherapeutic agents include but are not limited anti-fibrotic agents.

“Concomitant administration” of a known therapeutic agent with apharmaceutical composition of the present invention means administrationof the therapeutic agent and inhibitor agent at such time that both theknown therapeutic agent and the composition of the present inventionwill have a therapeutic effect. Such concomitant administration mayinvolve concurrent (i.e. at the same time), prior, or subsequentadministration of the drug with respect to the administration of acompound of the present invention. A person of ordinary skill in the artwould have no difficulty determining the appropriate timing, sequenceand dosages of administration for particular drugs and compositions ofthe present invention. Therapeutic agents contemplated for concomitantadministration according to the methods of the present invention includeany other agent for use in the treatment of fibrosis.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

The phrase “determining the treatment efficacy” and variants thereof caninclude any methods for determining that a treatment is providing abenefit to a subject. The term “treatment efficacy” and variants thereofare generally indicated by alleviation of one or more signs or symptomsassociated with the disease and can be readily determined by one skilledin the art. “Treatment efficacy” may also refer to the prevention oramelioration of signs and symptoms of toxicities typically associatedwith standard or non-standard treatments of a disease. Determination oftreatment efficacy is usually indication and disease specific and caninclude any methods known or available in the art for determining that atreatment is providing a beneficial effect to a patient. For example,evidence of treatment efficacy can include but is not limited toremission of the disease or indication. Further, treatment efficacy canalso include general improvements in the overall health of the subject,such as but not limited to enhancement of patient life quality, increasein predicted subject survival rate, decrease in depression or decreasein rate of recurrence of the indication (increase in remission time).(See, e.g., Physicians' Desk Reference (2010).)

DETAILED DESCRIPTION OF THE EMBODIMENTS

AXL, MER, Tyro3 and GAS6, as well as related pathways, have beendescribed in WO2011/091305, as well as U.S. application Ser. Nos.13/554,954 and 13/595,936; all of which are incorporated herein byreference in their entireties for all purposes.

In some embodiments, the methods of the present invention can be usedfor treatment, prevention or reduction of fibrosis, with an effectivedose of an inhibitor of a GAS6 or AXL associated signaling pathways.

In some embodiments, the inhibitor agent binds to two or more epitopeson a single GAS6 molecule. The two or more epitopes can include at leastone of the major and/or minor AXL, MER and/or Tyro3 binding site onGAS6. In some embodiments, the epitopes are separate or distinctepitopes. In some embodiments the epitopes overlap. In some embodiments,the epitopes do not overlap. In some embodiments, the epitopes areadjacent. In some embodiments, the epitopes are not adjacent. In someembodiments, the epitopes include the major and/or minor AXL, MER and/orTyro3 binding site on GAS6. These GAS6 epitopes of the presentinvention, and to which the inhibitor agents of the present inventionbind, can be located on one or more GAS6 molecules. In some embodiments,the epitopes are located on a single GAS6 molecule.

In some embodiments, the inhibitor agent is capable of binding to themajor and/or minor AXL, MER and/or Tyro3 binding sites on a single GAS6.In some embodiments, the inhibitor agent is capable of binding the majorAXL, MER and/or Tyro3 binding site of GAS6 and one or more additionalGAS6 epitopes. In other embodiments, the inhibitor agent is capable ofbinding to the AXL, MER and/or Tyro3 minor binding site on GAS6 and oneor more additional epitopes. In some other embodiments, the inhibitoragent is capable of binding two or more distinct epitopes on GAS6. Theadditional GAS6 epitopes can include any epitopes on GAS6 which lead toincreased affinity and/or increased avidity of the inhibitor agentbinding to GAS6 as compared to wild-type AXL, MER and/or Tyro3. In someembodiments, the AXL, MER and/or Tyro3 variant polypeptides of thepresent invention bind two epitopes on a single GAS6 molecule. In someembodiments, the two epitopes are the major and minor AXL, MER and/orTyro3 binding sites.

According to the invention, GAS6 receptors include AXL, MER and Tyro3.The inhibitor agents of the present invention can also in someembodiments antagonize the major and/or minor GAS6/receptor interaction.In some embodiments, the inhibitor agent is capable of antagonizing themajor and/or minor GAS6/receptor binding interaction. In otherembodiments, the inhibitor agent is capable of antagonizing the majorGAS6/receptor binding interaction (e.g., interfering with and/orinhibiting the major GAS6/receptor binding interaction). In someembodiments, the inhibitor agent is capable of antagonizing the minorGAS6/receptor binding interaction (e.g., interfering with and/orinhibiting the minor GAS6/receptor binding interaction).

Inhibitor agents can also include for example protein scaffolds (i.e.,smaller proteins that are capable of achieving comparable affinity andspecificity using molecular structures that can be for example one-tenththe size of full antibodies). The inhibitor agents can also includenon-antibody polypeptides. In some embodiments, the inhibitor agent is anon-antibody polypeptide. In some embodiments, the non-antibodypolypeptide can include but is not limited to peptibodies, darpins,avimers, adnectins, anticalins, affibodies, maxibodies, or other proteinstructural scaffold, or a combination thereof.

In some embodiments the inhibitor agent provided by the presentinvention is an AXL, MER and/or Tyro3 variant polypeptide, e.g., an AXL,MER and/or Tyro3 variant polypeptide that has a binding activity to GAS6that is substantially equal to or better than the binding activity of awild-type AXL, MER and/or Tyro3 polypeptide. In some embodiments of thepresent invention, the AXL, MER and/or Tyro3 variant polypeptides areutilized as therapeutic agents.

The AXL protein, with reference to the native sequence of SEQ ID NO: 1,comprises an immunoglobulin (Ig)-like domain from residues 27-128, asecond Ig-like domain from residues 139-222, fibronectin type 3 domainsfrom residues 225-332 and 333-427, intracellular domain from residues473-894 including tyrosine kinase domain. The tyrosine residues at 779,821 and 866 become autophosphorylated upon receptor dimerization andserve as docking sites for intracellular signaling molecules. The nativecleavage site to release the soluble form of the polypeptide lies atresidues 437-451.

For the purposes of the invention, a soluble form of AXL (soluble AXL,sAXL or sAXL polypeptide) includes both wild-type AXL and AXL variantpolypeptides and is the portion of the polypeptide that is sufficient tobind GAS6 at a recognizable affinity, e.g., high affinity, whichnormally lies between the signal sequence and the transmembrane domain,i.e. generally from about SEQ ID NO: 1 residue 19-437, but which maycomprise or consist essentially of a truncated version of from aboutresidue 19, 25, 30, 35, 40, 45, 50 to about residue 132, 450, 440, 430,420, 410, 400, 375, 350, to 321, e.g., residue 19-132. According to themethods of the present invention, SEQ ID NO:1 can be usedinterchangeably with amino acids 8-894 of SEQ ID NO: 1, both of whichrefer to the wild-type AXL sequence. In some embodiments, a soluble formof AXL lacks the transmembrane domain, and optionally the intracellulardomain.

In some embodiments, the inhibitor agent is an AXL variant polypeptidethat lacks the AXL transmembrane domain and has at least one mutationrelative to wild-type that increases affinity of the AXL polypeptidebinding to GAS6 as compared to wild-type GAS6.

The MER protein, with reference to the native SEQ ID NO:2, comprises animmunoglobulin (Ig)-like domain from residues 81-186, a second Ig-likedomain from residues 197-273, fibronectin type 3 domains from residues284-379 and 383-482, intracellular domain from residues 527-999including tyrosine kinase domain. The tyrosine residues at 749, 753, 754and 872 become autophosphorylated upon receptor dimerization and serveas docking sites for intracellular signaling molecules.

For the purposes of the invention, a soluble form of MER (sMER) is theportion of the polypeptide that is sufficient to bind GAS6 at arecognizable affinity, e.g., high affinity, which normally lies betweenthe signal sequence and the transmembrane domain, i.e. generally fromabout SEQ ID NO: 2 residue 21-526, but which may comprise or consistessentially of a truncated version In some embodiments, a soluble formof MER lacks the transmembrane domain (i.e., generally from about SEQ IDNO: 2 residue 506-526), and optionally the intracellular domain (i.e.,generally from about SEQ ID NO: 2 residue 527-999).

In some embodiments, the inhibitor agent is a soluble MER variantpolypeptide wherein said MER polypeptide lacks the MER transmembranedomain and has at least one mutation relative to wild-type thatincreases affinity of the MER polypeptide binding to GAS6 as compared towild-type MER.

The Tyro3 protein, with reference to the native SEQ ID NO:3, comprisesan immunoglobulin (Ig)-like domain from residues 41-128, a secondIg-like domain from residues 139-220, fibronectin type 3 domains fromresidues 225-317 and 322-413, intracellular domain from residues 451-890including tyrosine kinase domain. The tyrosine residues at 681, 685, 686and 804 become autophosphorylated upon receptor dimerization and serveas docking sites for intracellular signaling molecules.

For the purposes of the invention, a soluble form of Tyro3 (sTyro3) isthe portion of the Tyro3 polypeptide that is sufficient to bind GAS6 ata recognizable affinity, e.g., high affinity, which normally liesbetween the signal sequence and the transmembrane domain, i.e. generallyfrom about SEQ ID NO: 3 residue 41-450, but which may comprise orconsist essentially of a truncated version In some embodiments, asoluble form of AXL lacks the transmembrane domain (i.e., generally fromabout SEQ ID NO: 3 residue 430-450), and optionally the intracellulardomain (i.e., generally from about SEQ ID NO: 3 residue 451-890).

In some embodiments, the inhibitor agent is a soluble Tyro3 variantpolypeptide wherein said Tyro3 polypeptide lacks the Tyro3 transmembranedomain and has at least one mutation relative to wild-type Tyro3 thatincreases affinity of the Tyro3 polypeptide binding to GAS6 as comparedto wild-type Tyro3.

In some embodiments, the AXL, MET or Tyro3 variant polypeptide lacks theAXL, MET or Tyro3 transmembrane domain and is a soluble variantpolypeptide, e.g., polypeptides (sAXL, sMER or sTyro3 variantpolypeptide). In some embodiments, the AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 intracellular domain. In someembodiments, the inhibitor agent of the present invention inhibitsbinding between a wild-type AXL, MER and/or Tyro3 polypeptide and a GAS6protein in vivo or in vitro. In some embodiments, the AXL, MER or Tyro3variant polypeptide inhibits binding between a wild-type AXL, MER and/orTyro3 polypeptide and a GAS6 protein in vivo or in vitro.

The inhibitor agents of the present invention can also exhibit anenhanced or better pharmacokinetic profile. In some embodiments, theenhanced or better pharmacokinetic profile includes for example but isnot limited to a better absorption profile, better distribution profile,better metabolism profile, better excretion profile, better liberationprofile, increased half-life, decrease half-life, faster rate of action,longer duration of effect as compared to AXL, MER and/or Tyro3 wild-typepolypeptides which do not lack a transmembrane domain. One of skill inthe art would understand preferred pharmacokinetic profile parametersfor particular needs including for example treatment regimens, and howto appropriately implement such parameters in treatment regimens.

The wild-type AXL, MER and Tyro3 all contain two fibronectin domains. Insome embodiments, the AXL, MER and Tyro3 polypeptides of the inventionlack a functional fibronectin (FN) domain. Lacks or lacking a functionalfibronectin domain can include but is not limited to deletion of one orboth fibronectin domains and/or introducing mutations that inhibit,reduce or remove the functionality of one or both fibronectin domains,where such mutations can include for example but are not limited tosubstitution, deletion and insertion mutations. In some embodiments, thepolypeptides of the invention have fibronectin 1 (FN1) deleted,fibronectin 2 (FN2) deleted, or FN1 and FN2 both deleted. In someembodiments, the polypeptides of the invention have portions of FN1mutated and/or deleted, FN2 mutated and/or deleted, or FN1 and FN2mutated and/or deleted.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks afunctional AXL, MER or Tyro3 fibronectin (FN) domain. In someembodiments, the AXL, MER or Tyro3 variant polypeptide exhibitsincreased affinity of the polypeptide binding to GAS6 as compared towild-type AXL, MER and/or Tyro3. In some embodiments, the AXL, MER orTyro3 variant polypeptide lacks a functional fibronectin (FN) domainalso exhibits increased affinity of the polypeptide binding to GAS6 ascompared to wild-type AXL, MER and/or Tyro3.

In some embodiments, the lack of a functional fibronectin domain resultsin increased affinity of the AXL, MER or Tyro3 polypeptide binding toGAS6. In some embodiments, the lack of a functional fibronectin domainresults in an enhanced or better pharmacokinetic profile, including forexample but not limited to a better absorption profile, betterdistribution profile, better metabolism profile, better excretionprofile, better liberation profile, increased half-life, decreasedhalf-life, faster rate of action, longer duration of effect as comparedto other wild-type polypeptides or other polypeptides which do not lacka functional fibronectin domain. One of skill in the art wouldunderstand preferred pharmacokinetic profile parameters for particularneeds including for example treatment regimens, and how to appropriatelyimplement such parameters in treatment regimens.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain and has more than one Ig1 domain and exhibitsincreased affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6as compared to wild-type AXL, MER and/or Tyro3. In some embodiments, theAXL, MER or Tyro3 polypeptide has two Ig1 domains. In some embodiments,the AXL, MER or Tyro3 polypeptide has three Ig1 domains. In someembodiments, the AXL, MER or Tyro3 polypeptide has more than one Ig1domain and/or more than one Ig2 domain. In some embodiments, the AXL,MER or Tyro3 polypeptide has two Ig2 domains. In some embodiments, theAXL, MER or Tyro3 polypeptide has two Ig1 domains and 2 Ig2 domains. Insome embodiments, the AXL, MER or Tyro3 polypeptide includes for examplebut is not limited to one of the following Ig domain configurations, aswell as any combinations or variations thereof: Ig1; Ig1-Ig2; Ig1-Ig1;Ig1-Ig1-Ig1; Ig1-Ig2-Ig1; Ig1-Ig2-Ig1-Ig2.

In some embodiments, the AXL, MER or Tyro3 polypeptide also lacks theAXL, MER or Tyro3 transmembrane domain and/or exhibits increasedaffinity of the AXL, MER or Tyro3 polypeptide binding to GAS6. In someembodiments, the AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain, has more than one Ig1 domain, has more than oneIg2 domain and exhibits increased affinity of the AXL, MER or Tyro3polypeptide binding to GAS6 as compared to wild-type AXL, MER and/orTyro3.

In some embodiments, the AXL, MER or Tyro3 has the immunoglobulindomains connected directly to one another. In some embodiments, the AXL,MER or Tyro3 has the immunoglobulin domains connected indirectly, e.g.,through a linker molecule including for example any amino acid linkerknown in the art.

In some embodiments, the one or more AXL, MER or Tyro3 Ig1 and/or 1 ormore AXL, MER or Tyro3 Ig2 domains result in an enhanced or betterpharmacokinetic profile, including for example but not limited to abetter absorption profile, better distribution profile, bettermetabolism profile, better excretion profile, better liberation profile,increased half-life, decreased half-life, faster rate of action, longerduration of effect as compared to other wild-type polypeptides or otherpolypeptides which do not lack a functional fibronectin domain. One ofskill in the art would understand preferred pharmacokinetic profileparameters for particular needs including for example treatmentregimens, and how to appropriately implement such parameters intreatment regimens.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks theAXL, MER or Tyro3 transmembrane domain and is capable of binding two ormore epitopes on a single GAS6. In some embodiments, the AXL, MER orTyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembranedomain and is capable of binding both the major and minor AXL, MERand/or Tyro3 binding sites on a single GAS6. In some embodiments, thebinding of both the major and minor AXL, MER and/or Tyro3 binding issimultaneous. In some embodiments, the binding of both the major andminor AXL, MER and/or Tyro3 binding sites is simultaneous on a singleGAS6.

The present invention also provides AXL, MER or Tyro3 variantpolypeptides that do not bind two epitopes on a single GAS6 molecule.The present invention also provides AXL, MER or Tyro3 variantpolypeptides that do not bind two epitopes on a single GAS6 moleculesimultaneously. In some embodiments, the AXL, MER and/or Tyro3 variantpolypeptide is not capable of binding two epitopes on a single GAS6,this includes for example monomeric AXL, MER and/or Tyro3 variantpolypeptides. In some embodiments, the monomeric AXL, MER or Tyro3variant polypeptide comprises one Ig1 domain. In some embodiments, themonomeric AXL, MER and/or Tyro3 variant polypeptide is an Fc fusionpolypeptide that does not bind to more than one site on a single Gas6molecule simultaneously. In some embodiments, the monomeric AXL, MERand/or Tyro3 variant polypeptide that is not capable of binding twoepitopes on a single GAS6 comprises two AXL, MER and/or Tyro3 variantpolypeptides each of which are not capable of binding two epitopes on asingle GAS6 simultaneously. In some embodiments, the monomeric AXL, MERand/or Tyro3 variant polypeptide that is not capable of simultaneouslybinding two epitopes on a single GAS6 has one Ig1 domain. In someembodiments, the monomeric AXL, MER and/or Tyro3 variant polypeptidethat is not capable of simultaneously binding two epitopes on a singleGAS6 has an altered half-life when compared to AXL, MER and/or Tyro3variant polypeptides that are capable of binding two epitopes on asingle GAS6. In some embodiments, the polypeptide has one Ig1 domain andlacks a functional Ig2 domain. In some embodiments, the Ig1 domaincomprises amino acids 1-131 of AXL (SEQ ID NO:1). In some embodiments,the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide,wherein said soluble AXL, MER or Tyro3 variant polypeptide lacks theAXL, MER or Tyro3 transmembrane domain, has one Ig1 domain, lacks afunctional Ig2 domain and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3 variantpolypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. Insome embodiments, the polypeptide of any of the preceding claims,wherein the polypeptide is a soluble AXL, MER or Tyro3 variantpolypeptide, wherein said soluble AXL, MER or Tyro3 variant polypeptidelacks the AXL, MER or Tyro3 transmembrane domain, lacks a functionalfibronectin (FN) domain, has one Ig1 domain, lacks a functional Ig2domain and wherein said AXL, MER or Tyro3 variant polypeptide exhibitsincreased affinity of the AXL, MER or Tyro3 variant polypeptide bindingto GAS6 compared to wild-type AXL, MER or Tyro3.

The wild-type AXL, MER and Tyro3 all contain an Ig2 domain. In someembodiments, the AXL, MER and Tyro3 polypeptides of the invention lack afunctional Ig2 domain. Lacks or lacking a functional Ig2 domain caninclude but is not limited to deletion of the Ig2 domain and/orintroduction of mutations that inhibit, reduce or remove thefunctionality of the Ig2 domain, where such mutations can include forexample but are not limited to substitution, deletion and insertionmutations. In some embodiments, the polypeptides of the invention lack afunctional Ig2 domain. In some embodiments, the polypeptides of theinvention lack a functional Ig2 domain and have a wild-type AXL, MERand/or Tyro3 Ig1 domain. In some embodiments, the polypeptides of theinvention lack a functional Ig2 domain and have one or more mutations inthe Ig1 domain relative to the wild-type AXL, MER and/or Tyro3 Ig1domain.

In some embodiments, the AXL, MER and/or Tyro3 variant polypeptideincludes a linker. A wide variety of linkers are known in the art andany known linker can be employed with the methods of the presentinvention. In some embodiments, the AXL, MER or Tyro3 variantpolypeptide includes one or more linkers or linker units. In someembodiments, the linker is an amino acid linker, including an amino acidsequence of 2, 3, 4 or 5 amino acids which are different that thewild-type AXL, MER and/or Tyro3 sequences. In some embodiments, thelinker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more units. In someembodiments, the linker is (GLY)₄SER (SEQ ID NO: 4). In someembodiments, the linker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more(GLY)₄SER units (SEQ ID NO: 4). In some embodiments, the linker has 1,2, 3 or 5 (GLY)₄SER units (SEQ ID NO: 4). In some embodiments, thelinkers are between the AXL, MER or Tyro3 variant polypeptide and the Fcportion of a fusion polypeptide. In some embodiments, the linkers arebetween the AXL, MER or Tyro3 variant polypeptide and the Fc portion ofa fusion polypeptide and the AXL, MER or Tyro3 variant polypeptide lacksa functional fibronectin domain.

In some embodiments, AXL, MER and/or Tyro3 variant polypeptides of thepresent invention also include one or more amino acid modificationswithin the soluble form of wild-type AXL, MER and/or Tyro3, e.g., one ormore amino acid modifications that increase its affinity for GAS6.According to the present invention, amino acid modifications include anynaturally occurring or man-made amino acid modifications known or laterdiscovered in the field. In some embodiments, amino acid modificationsinclude any naturally occurring mutation, e.g., substitution, deletion,addition, insertion, etc. In some other embodiments, amino acidmodifications include replacing existing amino acid with another aminoacid, e.g., a conservative equivalent thereof. In yet some otherembodiments, amino acid modifications include replacing one or moreexisting amino acids with non-natural amino acids or inserting one ormore non-natural amino acids. In still some other embodiments, aminoacid modifications include at least 1, 2, 3, 4, 5, or 6 or 10 amino acidmutations or changes.

In some exemplary embodiments, one or more amino acid modifications canbe used to alter properties of the soluble form of AXL, MER and/or Tyro3e.g., affecting the stability, binding activity and/or specificity, etc.Techniques for in vitro mutagenesis of cloned genes are known.

In some embodiments, AXL variant polypeptides, including for examplesAXL variants, of the present invention include one or more amino acidmodifications within one or more regions of residue 18 to 130, residue10 to 135, residue 15 to 45, residue 60 to 65, residue 70 to 80, residue85 to 90, residue 91 to 99, residue 104 to 110, residue 111 to 120,residue 125 to 130, residue 19 to 437, residue 130 to 437, residue 19 to132, residue 21 to 132, residue 21 to 121, residue 26 to 132, or residue26 to 121 of wild-type AXL. In some other embodiments, AXL variantpolypeptides of the present invention include one or more amino acidmodifications within one or more regions of residue 20 to 130, residue37 to 124 or residue 141 to 212 of wild-type AXL. In yet some otherembodiments, variants of the present invention include one or more aminoacid modifications at one or more positions of position 19, 23, 26, 27,32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105,109, 112, 113, 116, 118, 127, or 129 of wild-type AXL.

In yet some other embodiments, AXL polypeptide variants of the presentinvention include one or more amino acid modifications including withoutany limitation 1) A19T, 2) T23M, 3) E26G, 4) E27G or E27K, 5) G32S, 6)N33S, 7) T38I, 8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E,14) V79M, 15) Q86R, 16) D87G, 17) D88N, 18) 190M or 190V, 19) V92A, V92Gor V92D, 20) 197R, 21) T98A or T98P, 22) T105M, 23) Q109R, 24) V112A,25) F113L, 26) H116R, 27) T118A, 28) G127R or G127E, and 29) E129K and acombination thereof.

In yet some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications at position 32,87, 92, or 127 of wild-type AXL (SEQ ID NO: 1) or a combination thereof,e.g., G32S; D87G; V92A and/or G127R. In yet some other embodiments, AXLpolypeptide variants of the present invention include one or more aminoacid modifications at position 26, 79, 92, 127 of wild-type AXL (SEQ IDNO: 1) or a combination thereof, e.g., E26G, V79M; V92A and/or G127E. Inyet some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications at position 32,87, 92, 127 and/or 72 of wild-type AXL or a combination thereof, e.g.,G32S; D87G; V92A; G127R and/or A72V. In yet some other embodiments, AXLvariant polypeptides of the present invention include one or more aminoacid modifications at position 87, 92 and/or 127 of wild-type AXL (SEQID NO: 1) or a combination thereof, e.g., D87G; V92A; and/or G127R. Inyet some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications at position 32,92, and/or 127 of wild-type AXL (SEQ ID NO: 1) or a combination thereof,e.g., G32S; V92A; and/or G127R. In yet some other embodiments, AXLvariant polypeptides of the present invention include one or more aminoacid modifications at position 32, 87 and/or 127 of wild-type AXL (SEQID NO: 1) or a combination thereof, e.g., G32S; D87G; and/or G127R. Inyet some other embodiments, AXL polypeptide variants of the presentinvention include one or more amino acid modifications at position 32,87 and/or 92 of wild-type AXL (SEQ ID NO: 1) or a combination thereof,e.g., G32S; D87G; and/or V92A. In yet some other embodiments, AXLvariant polypeptides of the present invention include one or more aminoacid modifications at position 26, 79, 92, 127 of wild-type AXL (SEQ IDNO: 1) or a combination thereof, e.g., E26G, V79M; V92A and/or G127E. Inyet some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications at position 87and 92 of wild-type AXL (SEQ ID NO: 1) or a combination thereof, e.g.,D87G and V92A. In yet some other embodiments, AXL variant polypeptidesof the present invention include at least one amino acid modification atposition 72 of wild-type AXL (SEQ ID NO: 1), e.g., A72V.

According to the present invention, the inhibitor agent can include butis not limited to a polypeptide, a polypeptide-carrier fusion, apolypeptide-Fc fusion, polypeptide-conjugate, a polypeptide-drugconjugate, an antibody, a bispecific antibody, an antibody-drugconjugate, an antibody fragment, an antibody-related structure, or acombination thereof.

The inhibitor agents of the present invention can include peptides orpolypeptides. The peptides and polypeptides of the present invention caninclude natural and/or synthetic polypeptides. Synthetic polypeptidesand methods of making synthetic polypeptides are well known in the artand any known methods for making synthetic polypeptides can be employedwith the methods of the present invention. In some embodiments, theinhibitor agent is a natural or synthetic polypeptide. In someembodiments, the inhibitor agent is a natural or syntheticpolypeptide-fusion. In some embodiments, the inhibitor agent is anatural or synthetic polypeptide-Fc fusion. In some embodiments thenatural or synthetic polypeptide-fusion is a fusion with another proteinstructural class or scaffold or a natural or syntheticpolypeptide-fusion with a polymer or hydrogel or related structure.

According to the present invention, the AXL, MER or Tyro3 variantpolypeptides of the present invention can be further modified, e.g.,joined to a wide variety of other oligopeptides or proteins for avariety of purposes. For instance, various post-translation orpost-expression modifications can be carried out with respect to AXL,MER or Tyro3 variant polypeptides of the present invention. For example,by employing the appropriate coding sequences, one may providefarnesylation or prenylation. In some embodiments, the AXL, MER or Tyro3variant polypeptides of the present invention can be PEGylated, wherethe polyethyleneoxy group provides for enhanced lifetime in the bloodstream. The AXL, MER or Tyro3 variant polypeptides of the presentinvention can also be combined with other proteins, such as the Fc of anIgG isotype, which can be complement binding. The inhibitor agents ofthe present invention can include polypeptide conjugates andantibody-conjugates. In some embodiments, the inhibitor agent is apolypeptide-conjugate or antibody-conjugate. In some embodiments, thepolypeptide conjugate is a drug conjugate. In some embodiments, thepeptide or polypeptide conjugate is an antibody-drug conjugates. In someembodiments, the polypeptide conjugate is a polymer conjugate. Polymersof the present invention include but are not limited to PEG,PEG-containing polymers, degradable polymers, biocompatible polymers,hydrogels, as well as other polymer structures that could be conjugatedto a polypeptide, and can include combinations thereof.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide of thepresent invention is a fusion protein, e.g., fused in frame with asecond polypeptide. In some embodiments, the second polypeptide iscapable of increasing the size of the fusion protein, e.g., so that thefusion protein will not be cleared from the circulation rapidly. In someother embodiments, the second polypeptide is part or whole of Fc region.In some other embodiments, the second polypeptide is any suitablepolypeptide that is substantially similar to Fc, e.g., providingincreased size and/or additional binding or interaction with Igmolecules. In some embodiments, the sAXL-Fc fusion molecule is a solublemolecule. In some embodiments, the sAXL-Fc fusion has enhanced affinitytoward GAS6. In some embodiments, the sAXL-Fc fusion is a solublemolecule that has enhanced affinity toward GAS6. In some otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size and/oradditional binding or interaction with Ig molecules. In yet some otherembodiments, the second polypeptide is part or whole of an albuminprotein, e.g., a human serum albumin protein. In some embodiments, thesecond polypeptide is a protein or peptide that binds to albumin.

In some other embodiments, the second polypeptide is useful for handlingthe AXL, MER or Tyro3 variant polypeptides, e.g., purification of AXL,MER or Tyro3 variant polypeptides or for increasing its stability invitro or in vivo. For example, AXL, MER or Tyro3 variant polypeptides ofthe present invention can be combined with parts of the constant domainof immunoglobulins (IgG), resulting in chimeric or fusion polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. EP A 394,827; Traunecker et al., Nature, 331:84-86, 1988. Fusion proteins having disulfide-linked dimeric structures(due to the IgG) can also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

In yet some other embodiments, the second polypeptide is a markersequence, such as a peptide which facilitates purification of the fusedpolypeptide. For example, the marker amino acid sequence can be ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. Wilson et al., Cell 37: 767, 1984.

In still some other embodiments, the second polypeptide is an entityuseful for improving the characteristics of AXL, MER or Tyro3polypeptide variants of the present invention. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the AXL,MER or Tyro3 polypeptide variants of the present invention to facilitatepurification and subsequently removed prior to final preparation of thepolypeptide. The addition of peptide moieties to facilitate handling ofpolypeptides are familiar and routine techniques in the art.

In still yet some embodiments, AXL, MER or Tyro3 variant polypeptides ofthe present invention have a binding activity to GAS6 that is at leastequal or better than the wild-type AXL, MER or Tyro3. In some otherembodiments, AXL, MER or Tyro3 variant polypeptides of the presentinvention has a binding activity or affinity to GAS6 that is at least1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold greater than that ofthe wild-type AXL, MER or Tyro3. In some other embodiments, AXL, MER orTyro3 polypeptide variant of the present invention has a bindingactivity or affinity to GAS6 of at least about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸ or1×10⁻⁹ M, 1×10⁻¹⁰M, 1×10⁻¹¹M or 1×10⁻¹²M. In yet some other embodiments,sAXL polypeptides of the present invention is capable of inhibiting,inhibit or compete with wild-type AXL binding to GAS6 either in vivo, invitro or both. In yet some other embodiments, sAXL polypeptides of thepresent invention inhibit or compete with the binding of AXL S6-1, AXLS6-2, and/or AXL S6-5 (as described in WO2011/091305). In yet some otherembodiments, sAXL polypeptides of the present invention inhibit orcompete with the binding of any sAXL variant as described inWO2011/091305.

The inhibitor agents of the present invention bind to GAS6 withincreased affinity. In some embodiments, the AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3polypeptide binding to GAS6 as compared to wild-type AXL, MER or Tyro3.In some embodiments, AXL, MER or Tyro3 variant polypeptide exhibits anaffinity to GAS6 that is at least about 5-fold stronger, at least about10-fold stronger or at least about 20-fold stronger, 50-fold stronger,100-fold stronger or at least 200-fold stronger, etc. than the affinityof the wild-type AXL, MER or Tyro3 polypeptide. In some embodiments, thesoluble AXL has a about a 115-fold stronger affinity to GAS6 than theaffinity of the wild-type AXL polypeptide.

The ability of a molecule to bind to GAS6 can be determined, forexample, by the ability of the putative ligand to bind to GAS6 coated onan assay plate. In one embodiment, the binding activity of AXL, MER orTyro3 variant polypeptides of the present invention to a GAS6 can beassayed by either immobilizing the ligand, e.g., GAS6 or the AXL, MER orTyro3 variant polypeptides. For example, the assay can includeimmobilizing GAS6 fused to a His tag onto Ni-activated NTA resin beads.Agents can be added in an appropriate buffer and the beads incubated fora period of time at a given temperature. After washes to remove unboundmaterial, the bound protein can be released with, for example, SDS,buffers with a high pH, and the like and analyzed.

In still yet other embodiments, AXL, MER or Tyro3 variant polypeptidesof the present invention has a better thermal stability than the thermalstability of a wild-type AXL. In some embodiments, the meltingtemperature of AXL, MER or Tyro3 variant polypeptides of the presentinvention is at least 5° C., 10° C., 15° C., or 20° C. higher than themelting temperature of a wild-type AXL.

According to the present invention, AXL, MER or Tyro3 variantpolypeptides of the present invention can also include one or moremodifications that do not alter primary sequences of the AXL, MER orTyro3 variant polypeptides of the present invention. For example, suchmodifications can include chemical derivatization of polypeptides, e.g.,acetylation, amidation, carboxylation, etc. Such modifications can alsoinclude modifications of glycosylation, e.g. those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; e.g. by exposing thepolypeptide to enzymes which affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. In some embodiments, AXL, MERor Tyro3 polypeptide variants of the present invention include AXL, MERor Tyro3 variant polypeptides having phosphorylated amino acid residues,e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

In some other embodiments, AXL, MER or Tyro3 variant polypeptides of thepresent invention include AXL, MER or Tyro3 variant polypeptides furthermodified to improve their resistance to proteolytic degradation or tooptimize solubility properties or to render them more suitable as atherapeutic agent. For example, AXL, MER or Tyro3 polypeptide variantsof the present invention further include analogs of AXL, MER or Tyro3variant polypeptides containing residues other than naturally occurringL-amino acids, e.g. D-amino acids or non-naturally occurring syntheticamino acids. D-amino acids may be substituted for some or all of theamino acid residues.

In yet some other embodiments, AXL, MER or Tyro3 variant polypeptides ofthe present invention include at least two same or different AXL, MER orTyro3 variant polypeptides linked covalently or non-covalently. Forexample, in some embodiments, AXL, MER or Tyro3 polypeptide variants ofthe present invention include two, three, four, five, or six same ordifferent AXL, MER or Tyro3 variant polypeptides linked covalently,e.g., so that they will have the appropriate size, but avoiding unwantedaggregation.

According to the present invention, AXL, MER or Tyro3 variantpolypeptides of the present invention can be produced by any suitablemeans known or later discovered in the field, e.g., produced fromeukaryotic or prokaryotic cells, synthesized in vitro, etc. Where theprotein is produced by prokaryotic cells, it may be further processed byunfolding, e.g. heat denaturation, DTT reduction, etc. and may befurther refolded, using methods known in the art.

The AXL, MER or Tyro3 variant polypeptides may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman,etc. By using synthesizers, naturally occurring amino acids may besubstituted with unnatural amino acids. The particular sequence and themanner of preparation will be determined by convenience, economics,purity required, and the like.

The AXL, MER or Tyro3 variant polypeptides may also be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique. For the most part, the compositions which are used willcomprise at least 20% by weight of the desired product, more usually atleast about 75% by weight, preferably at least about 95% by weight, andfor therapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized. One of skill in the art can readily utilize well-knowncodon usage tables and synthetic methods to provide a suitable codingsequence for any of the polypeptides of the invention. Direct chemicalsynthesis methods include, for example, the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. While chemical synthesis of DNA isoften limited to sequences of about 100 bases, longer sequences can beobtained by the ligation of shorter sequences. Alternatively,subsequences may be cloned and the appropriate subsequences cleavedusing appropriate restriction enzymes.

The nucleic acids may be isolated and obtained in substantial purity.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome. The nucleic acids of the invention can be provided as alinear molecule or within a circular molecule, and can be providedwithin autonomously replicating molecules (vectors) or within moleculeswithout replication sequences. Expression of the nucleic acids can beregulated by their own or by other regulatory sequences known in theart. The nucleic acids of the invention can be introduced into suitablehost cells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

In some embodiments, the present invention provides expression vectorsfor in vitro or in vivo expression of one or more AXL, MER and/or Tyro3polypeptide variants of the present invention, either constitutively orunder one or more regulatory elements. In some embodiments, the presentinvention provides a cell population comprising one or more expressionvectors for expressing AXL, MER and/or Tyro3 polypeptide variants of thepresent invention, either constitutively or under one or more regulatoryelements.

According to the present invention, the AXL, MER or Tyro3 variantpolypeptides can be provided in pharmaceutical compositions suitable fortherapeutic use, e.g., for human treatment. In some embodiments,pharmaceutical compositions of the present invention include one or moretherapeutic entities of the present invention, e.g., AXL polypeptidevariants or pharmaceutically acceptable salts, esters or solvatesthereof or any prodrug thereof. In some other embodiments,pharmaceutical compositions of the present invention include one or moretherapeutic entities of the present invention in combination withanother therapeutic agent, e.g., another agent for treatment offibrosis.

Pirfenidone, marketed under the names Esbriet and Pirespa, is the firsttargeted antifibrotic drug to be approved for the treatment of IPF inEurope and Japan Although its exact mechanism of action remains unclear,pirfenidone is believed to attenuate fibroblast proliferation and theproduction by activated myofibroblasts of fibrosis-associated mediatorsand ECM components. Pirfenidone has also shown efficacy in preclinicalmodels of liver fibrosis, renal fibrosis, hypertrophic cardiomyopathyand radiation-induced fibrosis, suggesting that it may have broadantifibrotic activity. Therapeutic antibodies to TGF-β1, a key cytokineinvolved in the activation of myofibroblasts; CTGF, a matrix-associated,heparin-binding protein that mirrors the profibrotic activity of TGF-βon fibroblasts; and integrin α_(v)β₆, which is responsible for theactivation of constitutively expressed latent TGF-β, are also beinginvestigated for their antifibrotic activity. A humanized monoclonalantibody to α_(v)β₆ developed by Stromedix and Biogen Idec is beinginvestigated as a treatment for interstitial fibrosis and tubularatrophy in kidney-transplant recipients and as a therapy for IPF.Genzyme is also exploring a humanized pan-TGF-β inhibitor (fresolimumab)as a treatment for patients with early-stage diffuse systemic sclerosis,focal segmental glomerulosclerosis, IPF and myelofibrosis, andantibodies and antisense drugs targeting CTGF are being investigated inIPF and scar-revision surgery. Antagonists of the lysophosphatidicacid-1 receptor, a growth factor that induces CTGF and TGF-β1expression, are being considered as treatments for kidney fibrosis, IPFand systemic sclerosis. Bone morphogenetic protein-7 has also beenidentified as a potential therapeutic agent for chronic renal injurybecause it can counteract TGF-β1-induced EMT. An antagonist of theendothelin receptor, which promotes myofibroblast contraction andmigration, is being explored in cardiovascular disease and IPF. Ahumanized monoclonal antibody targeting lysyl oxidase-like-2, an enzymethat catalyzes the cross-linking of collagen, is being explored byGilead Sciences as a treatment for cardiac fibrosis, IPF and liverfibrosis. Other matrix assembly proteins, such as prolyl hydroxylases,are being investigated preclinically for antifibrotic activity.Bortezomib, a proteasomal inhibitor, inhibits TGF-β1 signaling in vitroand has been shown to protect mice from bleomycin-induced skin and lungfibrosis. It also induces apoptosis of hepatic stellate cells.Consequently, bortezomib may prove efficacious for diseases in whichTGF-β1, ER stress and activated myofibroblasts have been identified askey pathogenic mediators. Studies are also under way to examine whethera serine/threonine protein kinase inhibitor reduces the number ofcirculating fibrocytes in individuals with IPF.

The T_(H)2-associated cytokine IL-13 has emerged as a key driver ofinfection and allergen-driven fibrosis. IL-13 and its receptors havealso been detected at high levels in the lungs and blood of patientswith IPF. Because a growing number of chronic fibrotic diseases arecharacterized by the excess production of IL-13 and/or increasedexpression of IL-13-inducible genes, many individuals with fibrosismight benefit from the neutralization of IL-13.

In yet some other embodiments, pharmaceutical compositions of thepresent invention include one or more therapeutic entities of thepresent invention in combination with a pharmaceutically acceptableexcipient.

In still some other embodiments, therapeutic entities of the presentinvention are often administered as pharmaceutical compositionscomprising an active therapeutic agent, i.e., and a variety of otherpharmaceutically acceptable components. (See Remington's PharmaceuticalScience, 15^(th) ed., Mack Publishing Company, Easton, Pa., 1980). Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In still some other embodiments, pharmaceutical compositions of thepresent invention can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

In yet other embodiments, methods of the present invention includeadministering to a subject in need of treatment a therapeuticallyeffective amount or an effective dose of a therapeutic entity (e.g.,inhibitor agent) of the present invention, e.g., an inhibitor of AXL,MER and/or Tyro3 activity or GAS6 activity or an inhibitor ofinteraction between AXL, MER and/or Tyro3 and GAS6. In some embodiments,effective doses of the therapeutic entity of the present inventiondescribed herein vary depending upon many different factors, includingmeans of administration, target site, physiological state of thepatient, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human but nonhuman mammals includingtransgenic mammals can also be treated. Treatment dosages need to betitrated to optimize safety and efficacy.

In some embodiments, the dosage may range from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per every two weeks or once a month or once every 3to 6 months. Therapeutic entities of the present invention are usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, therapeutic entities of the present inventioncan be administered as a sustained release formulation, in which caseless frequent administration is required. Dosage and frequency varydepending on the half-life of the polypeptide in the patient.

In prophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

In still yet some other embodiments, for prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of a disease or condition in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presenting during development ofthe disease.

In still yet some other embodiments, for therapeutic applications,therapeutic entities of the present invention are administered to apatient suspected of, or already suffering from such a disease in anamount sufficient to cure, or at least partially arrest, the symptoms ofthe disease (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient response has been achieved.

According to the present invention, compositions can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal or intramuscular means. Themost typical route of administration is intravenous although otherroutes can be equally effective.

For parenteral administration, compositions of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water, oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies and/or polypeptides can be administered in the form of adepot injection or implant preparation which can be formulated in such amanner as to permit a sustained release of the active ingredient. Anexemplary composition comprises polypeptide at 1 mg/mL, formulated inaqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mML-arginine, 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or NaOH.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998.Co-administration can be achieved by using the components as a mixtureor as linked molecules obtained by chemical crosslinking or expressionas a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24,1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration. Preferably, a therapeutically effective dose willprovide therapeutic benefit without causing substantial toxicity.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch. 1).

Also within the scope of the invention are kits comprising thecompositions (e.g., AXL, MER or Tyro3 variant polypeptides andformulations thereof) of the invention and instructions for use. The kitcan further contain a least one additional reagent. Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. It is alsounderstood that the terminology used herein is for the purposes ofdescribing particular embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor only and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the appended claims.

EXPERIMENTAL

Experimental Methods and Procedures

Cell Culture. LM-P tumor cells (Clin Cancer Res 2010 Jul. 15; 16(14):3684-3695) were maintained in vitro as a monolayer culture in DMEMmedium supplemented with 10% heat inactivated fetal calf serum, 100 U/mlpenicillin and 100 μg/ml streptomycin, and L-glutamine (2 mM) at 37° C.in an atmosphere of 5% CO₂ in air. The tumor cells were routinelysubcultured twice weekly by trypsin-EDTA treatment. Cells growing in anexponential growth phase were harvested and counted using a BeckmanCoulter particle counter prior to tumor inoculation.

Tumor Inoculation. Each mouse was inoculated subcutaneously on the rightflank with PDA1-1 tumor cells (1×10⁶) in 0.1 ml of sterile saline fortumor development. Subcutaneous tumors were grown for two-three weeks.To establish orthotopic tumors, mice harboring the subcutaneous tumorswere sacrificed and the tumors were isolated and cut into small 3-4 mmfragments. Laparotomies were performed and a tumor fragment was securedto the tail of the pancreas using resorbable sutures. Afterimplantation, the pancreas was returned to the peritoneal cavity and theincision was closed. Mice received daily injections of carprofen on theday of implantation and on each of the three days post-op for painmanagement.

At day four post-surgery, mice were randomly divided into four groupsconsisting of 10 or 14 animals. The testing articles were administratedto the mice according to the predetermined regimen shown below.

Compounds Preparation Concentration Storage High affinity AXL 0.2 μmfilter sterilized 1 mg/ml 4° C. variant polypeptide in optimizedformulation Gemcitabine 0.2 μm filter sterilized 2 mg/ml Room in salinetemp

Masson Trichrome staining. Primary tumor tissue was obtained from eachmouse upon sacrifice, and was fixed in in 10% formalin. Fixed tissue wasmounted and the amount of collagen present was visualized by MassonTrichrome staining. Staining was performed according the manufacturer'sprotocol (American MasterTech, Lodi, Calif.).

For each tissue section, at least two fields of view were scored from0-4, with: 0 having no fibrosis; 1<20% collagen staining; 2 20-40%collagen staining; 3 40-60% collagen staining; 4>60% collagen staining.Average scores for each tissue section were calculated and reported.

All the procedures related to animal handling, care, and treatment inthis study were performed according to guidelines approved by theInstitutional Animal Care and Use Committee (IACUC) of StanfordUniversity following the guidance of the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC). At the time of routinemonitoring, the animals were checked for any effects of tumor growth onnormal behavior such as mobility, food and water consumption (by lookingonly), body weight gain/loss, eye/hair matting and any other abnormaleffect. Death and observed clinical signs were recorded on the basis ofthe numbers of animals within each subset.

RESULTS

Tumor fibrosis. Amount of fibrosis in primary tumor sections, asassessed by Masson Trichrome staining is shown in FIG. 1. Fibrotictissue is labeled by the blue stain in the representative images on theleft. The averaged, quantified labeling is reported in the graph on theright.

Treatment with high affinity AXL variant polypeptide decreased fibrosiswithin primary tumor tissue, as assessed by collagen content throughMasson Trichrome staining.

What is claimed is:
 1. A method of reducing fibrosis associated withpancreatic cancer, the method comprising: detecting the presence offibrosis in the pancreatic cancer, and then administering to a humanpatient with pancreatic cancer-associated fibrosis an inhibitor of GAS6,wherein the inhibitor is a soluble AXL variant polypeptide, wherein saidsoluble AXL variant polypeptide: lacks the AXL transmembrane domain,lacks a functional fibronectin (FN) domain, has an Ig1 domain, and anIg2 domain, and comprises a set of amino add substitutions relative toSEQ ID NO:1 selected from Gly32Ser, Asp87Gly, Val92Ala, and Gly127Arg orfrom Gly32Ser, Ala72Val, Asp87Gly, Val92Ala, and Gly127Arg; to therebyreduce the pancreatic cancer-associated fibrosis.
 2. The method of claim1 wherein the patient has been treated with gemcitabine.
 3. The methodof claim 1 wherein the soluble AXL variant polypeptide further comprisesan Fc domain linked to the AXL variant polypeptide by a linkercomprising from 1 to 5 (GLS)₄SER (SEQ ID NO:10) units.
 4. The method ofclaim 1 wherein the soluble AXL variant polypeptide further comprises aconjugated polymer selected from polyethylene glycol (PEG), aPEG-containing polymer, a degradable polymer, a biocompatible polymer,or a hydrogel.